Plants are sources of drugs and other useful materials. These materials are the products of plant metabolism, which is the total collection of biochemical pathways within a plant. Genes encode the enzymes that function in plant metabolic pathways and regulatory genes, in turn, control the activity of metabolic pathways by directing the expression of entire sets of enzyme-encoding genes specific to a given pathway. It follows that methods to identify any genes that govern the production of such plant metabolites are critical to enable the manipulation of pathways for elevating product yield and for increasing the diversity of these substances. The present invention provides methods to identify, in a relatively efficient and reliable manner, plant-genetic material required for production of metabolites which would ordinarily be difficult to identify. The invention is the realization of the successful merging of two distinct methodologies: activation-tagging mutagenesis and high-throughput screening. The present unique combination of these methodologies results in the ability to assign metabolic functionality to plant genes involved in the production of biologically active molecules and to create a means of compound discovery based on the genetic capabilities of a plant or group of plants.
This invention is therefore based on the following technical developments: 1) methodology which enables propagation and maintenance of mutagenized “microcallus” material in a “library” format in such a way as to permit large scale screening; 2) procedures to nondestructively sample the microcallus; 3) methodology to screen the microcallus sample in a miniaturized high throughput pharmacological screen.
Therefore, the invention is in the field of plant molecular biology, in particular, genetic methods for production and identification of useful compounds.
Identification of plant genes via activation mutagenesis has been used with success previous to the present invention. This technique involves incorporation of enhancer sequences from a plant viral promoter at random places into the plant genome via Agrobacterium mediated T-DNA transfer. When applied to single plant cells in culture, the resulting mutants are identified by positive selection, and the gene(s) in the vicinity of the T-DNA insert is cloned. However, elucidation of the effects of mutations in regulatory regions, or downstream effects from a mutagenized gene, are very difficult in cases where no positive selection schemes can be employed. Even with classical activation mutagenesis methods, Walden et al. conclude “this process is involved, labor-intensive and can only be effectively carried out with relatively limited numbers of segregating individuals.” Walden et al., Methods in Cell Biology, 49:455-469 (1995).
High-throughput screening has also been utilized in the pharmaceutical field.
Citation of the above document is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant and does not constitute any admission as to the accuracy of the dates or contents of these documents.